Project Summary Neuroblastoma (NB) is a pediatric cancer of the developing peripheral sympathetic nervous system and accounts for 15% of all childhood cancer deaths. A large majority of high-risk NB patients initially respond to chemotherapy but 60% will face therapy-resistant relapses. Current therapies for these patients are highly toxic and lack efficacy, indicating an urgent unmet need for new treatment options. Recently, whole genome sequencing analysis of paired primary and relapse tumors revealed that 78% of relapsed tumors had mutations activating the RAS-MAPK pathway (N=23). Aberrant signaling in relapsed tumors was shown to stem from acquired mutations and copy number variations in MAPK-related genes, including RAS, ALK, NF1, and PTPN11. Subsequent in vitro studies showed that 11/18 neuroblastoma cell lines had RAS-MAPK-activating mutations and were sensitive to inhibition of the downstream enzymes MEK1 and MEK2. Although MEK1/2 inhibitors (MEKi) appear efficacious in preclinical testing, targeting this critical pathway is notoriously difficult because resistance is common. I hypothesize that targeting MEK1/2 in combination with proteins conferring resistance to MEK-inhibition will provide therapeutic benefit in preclinical models of neuroblastoma. Current literature has implicated the Yap-Hippo pathway in RAS-MAPK resistance to MEKi therapies in melanoma and NSCLC. In Aim 1A, I will utilize lentiviral CRISPR-CAS9 genome editing to knock down YAP protein expression to observe effects on proliferation, survival, and cellular signaling with and without MEK inhibition. In Aim 1B, I will perform lentiviral-mediated overexpression of an unphosphorylated form of the YAP protein to investigate enhanced resistance to MEK inhibition using cell-based assays. To identify alternative resistance to MEKi therapy, I will study kinase drug targets obtained from a screen assessing cell viability in the presence of a MEK inhibitor and a protein kinase siRNA library in four RAS-MAPK-mutated cell lines. The screen two hits (KSR and MAP3K3) were common among three and 12 hits were common among two cell lines. In Aim 2A, I will utilize CRISPR-Cas9 knockdown studies of candidate kinase genes to functionally characterize synthetic lethality and resistance mechanisms in RAS-MAPK-mutated cell lines. In Aim 2B, I will prioritize validated kinase targets based on available pharmacological inhibitors and characterize in vitro and in vivo drug efficacy in combination with a MEK inhibitor. These studies will yield new insights into the cellular processes conferring resistance to RAS-MAPK-inhibition in neuroblastoma. This understanding will address a clinical need for novel combination therapies for relapsed patients with aberrant RAS-MAPK signaling and contribute to a deeper understanding of cancer signaling responses to MEK inhibition. ! !